This invention relates to biochemistry and medicine, and involves zeaxanthin and other carotenoids.
Zeaxanthin (occasionally abbreviated herein as ZX) is a particular carotenoid compound that is naturally present in the “macula”, a small yellow-pigmented region at the center of the retinas of humans and other mammals. As used herein, the term “zeaxanthin” includes any or all of the three stereoisomers of zeaxanthin, which are often designated as the 3R-3′R isomer (also referred to as the R—R isomer, for convenience), the S-R isomer (also known as meso-zeaxanthin), and the 3S-3′S isomer. In general, the R—R isomer is believed to be preferable, and it is the isomer generated by the Flavobacterium multivorum bacterial strains described below.
Abnormally low concentrations of zeaxanthin in the macula have been correlated with a retinal disease called “macular degeneration”. Since this disease usually manifests late in life, it is also called “age-related macular degeneration”, abbreviated as AMD (or occasionally as ARMD). To distinguish macular degeneration from several other “MD” abbreviations, “AMD” is the most commonly used acronym and abbreviation for macular degeneration.
In macular degeneration, the macula (which, as noted above, is at the center of the retina) becomes unable to function properly. This causes the person to become unable to distinguish things that are in the center of his or her field of vision. If macular degeneration continues to grow worse, it often leaves its victims effectively blind. Macular degeneration is a leading cause of blindness among the elderly.
Since zeaxanthin (like other carotenoids) is not synthesized inside any mammalian body, it must be ingested from food sources. This can pose problems, since it is present in most foods only at extremely low quantities.
It has been suspected for some years that nutritional supplements or pharmaceutical preparations containing ZX might be able to help prevent, treat, or reduce the risk or severity of macular degeneration. However, no vitamin-type (“nutraceutical”) supplements have been available with anything more than very small quantities of ZX. This is due to the very low concentration of ZX in most plant sources, and to the extremely high costs and difficulties in chemically synthesizing and purifying the 3R-3′R stereoisomer of ZX.
Five U.S. patents have been issued on ZX formulations generated by fermenting a bacterial line that was discovered to synthesize the 3R-3′R stereoisomer of ZX. The parent strain of those bacteria, known as Flavobacterium multivorum, was deposited with the American Type Culture Collection, and was given ATCC accession number 55238; however, because of changes in classification nomenclature, that strain is referred to in the ATCC catalog as Sphingobacterium multivorum.
The first two such patents (U.S. Pat. No. 5,308,759, Gierhart 1994, and U.S. Pat. No. 5,427,783, Gierhart 1995) relate to the use of ZX as an additive in feeds for poultry and fish. In that mode of usage, it causes the skin and egg yolks of poultry, and the meat of salmon and certain other fish, to turn a darker color, which is more appealing to consumers. Those types of feed additives typically take the form of a cell paste or other biomass, containing the remains of killed cells, without using extensive processing to extract or purify the zeaxanthin they contain.
The other three patents, all of which relate to medical use in humans, are U.S. Pat. No. 5,747,544 (Garnett et al 1998), which discloses a method of using the 3R-3′R stereoisomer of ZX to treat or prevent macular degeneration; U.S. Pat. No. 5,827,652 (Garnett et al 1998), which discloses orally ingestible formulations; and U.S. Pat. No. 5,854,015 (Garnett et al 1998), which discloses a method of making the purified 3R-3′R stereoisomer, using bacterial fermentation. Additional information on zeaxanthin, and on macular degeneration, is contained in these three U.S. patents, and the contents and teachings of those patents are incorporated herein by reference, as though fully set forth herein.
One of the current inventors herein, Luis Guerra-Santos, was involved in that development work, and is a coinventor of the three patents on ZX formulations for human use. This current invention arose out of subsequent research and development efforts which continued after the filing of those three applications.
It also should be recognized that a purely chemical method for synthesizing the R—R stereoisomer of zeaxanthin apparently has been recently been created, by Hoffman-LaRoche. That chemically synthesized version of R—R zeaxanthin is discussed in certain documents which have been submitted by Hoffman-LaRoche to the U.S. Food and Drug Administration, and which are accessible to the public via the Internet, in the FDA website.
It is assumed and believed that the processing steps disclosed herein can be tested on any type of synthetic, fermented, or other zeaxanthin (or other carotenoid) preparation having with average particle sizes larger than a micron in diameter, using no more than routine experimentation, to determine whether these steps can be used to create a “nanoparticle” zeaxanthin or other carotenoid preparation.
As used herein, the term “nanoparticle” is used to refer to a preparation of zeaxanthin (or other carotenoid) having particle sizes that are less than 1 micron in diameter, on average. Since a micron is one-millionth of a meter (equal to one-thousandth of a millimeter), and a nanometer is one billionth of a meter (or one-thousandth of a micron), this is comparable to saying that a “nanoparticle” preparation has particle sizes that average 999 nanometers, or less, in diameter. The term “sub-micron” formulation can be used interchangeably with “nanoparticle” formulation, since both terms imply that the particles have average diameters less than a micron.
The manufacturing processes disclosed herein offer a cost-effective way to create zeaxanthin preparations which consistently fall within the definition of “nanoparticle” or “sub-micron” formulations. Although it is presumed that these “nanoparticle” formulations will offer the highest possible bioavailability, for a given weight of zeaxanthin, it should be recognized that the final choice of a desired particle size range, for large-scale commercial quantities of zeaxanthin, will depend on economics, rather than on a rigid goal of smallest possible particles, and highest possible bioavailability, regardless of cost. It may turn out that the most cost-effective manufacturing process, which generates the highest level of total retinal tissue deposition among users for the lowest total manufacturing cost, may involve zeaxanthin preparations which have average diameters somewhat higher than 1 micron, and which therefore is not a true “nanoparticle” formulation.
Accordingly, the term “microcrystalline” as used herein is somewhat broader, and includes both (i) nanoparticle carotenoid formulations, and (ii) carotenoid formulations having average particle diameters of about 10 microns or less, and preferably having average particle diameters of about 4 microns or less.
By comparison, carotenoid formulations having average particle diameters larger than about 10 microns are referred to herein as “coarse-grained” formulations. Those are deemed to be the relevant prior art herein, and the goal of this invention is to provide carotenoid formulations with significantly smaller average particle sizes (and significantly better bioavailability) than coarse-grained carotenoid formulations.
Microcrystalline zeaxanthin-in-oil formulations, which can be prepared as disclosed herein (and which include nanoparticle formulations, as one subset of the microcrystalline size range) have substantially smaller average particle sizes than the prior zeaxanthin formulations that were prepared as described in U.S. Pat. Nos. 5,747,544; 5,827,652; and 5,854,015, as cited above. Since microcrystalline zeaxanthin formulations have substantially smaller particle sizes and better bioavailability than the coarse-grain formulations previously disclosed, they offer a substantial improvement over the prior known formulations, and are useful as such.
The phrases “zeaxanthin-in-oil” and “carotenoid-in-oil” are important herein, and are intended to focus upon a significant distinction between this invention, and the prior art. Various methods have been previously disclosed for preparing carotenoids (including zeaxanthin) in either of two different forms.
One form is usually referred to as a powder, “dry powder”, or “coldwater dispersible powder”. These types of powdered carotenoid preparations are disclosed in various publications, such as U.S. Pat. No. 5,968,251 (Auweter et al, 1999, assigned to BASF Aktiengesellschaft). These preparations are manufactured, shipped, and stored in bulk, in powdered form. Before being packaged for retail sale, this type of powder typically is loaded into a conventional capsule (which may be made of an enteric material that will not dissolve until the capsule enters the intestines; this protects the carotenoid from acidity in the stomach). Alternately, a carotenoid powder may be mixed with a binder compound and then compressed into tablet form; if desired, such tablets may be coated with a thin layer of an enteric coating, to create a convenient dosage form commonly known as “caplets”.
The other major class of carotenoid preparations in the prior art involves non-homogenous suspensions of oily droplets in aqueous phases. Depending on which types of compounds are used to sustain a non-homogenous suspension in a stable form (to prevent the oily droplets from coalescing and aggregating together, which is highly undesirable), these suspensions are usually referred to by terms such as emulsions, colloids, liposomes, micelles, etc.
By contrast, the methods and mixtures disclosed herein describe microcrystalline zeaxanthin (or other carotenoid) formulations which are suspended directly in an edible oily carrier, such as a vegetable oil. It is believed that this methodology can provide significant benefits when scaled up to commercial quantities, by combining microcrystalline particles sizes with stable yet inexpensive liquid carriers that offer several advantages. As examples of such advantages, these types of oily liquid carriers can: (i) protect the microcrystalline particles against oxidation, (ii) allow the use of relatively simple fluid-handing equipment and techniques, (iii) facilitate loading of the product into soft-gel capsules; and, (iv) eliminate or reduce the need for expensive specialized compounds that can effectively coat the carotenoid molecules, in a manner which will prevent them from clumping or coalescing.
In addition, the methods and carriers disclosed herein are believed to allow less harsh, less expensive, and easier-to-control processing steps, compared to the processing steps used to create dry powders or non-homogenous suspensions.
Also, the methods used to create the carotenoid-in-oil preparations disclosed herein are believed to pose lower risks of converting desirable isomer(s) or stereoisomer(s) of certain carotenoids (such as the 3R,3′R stereoisomer of zeaxanthin, or certain “cis” or “trans” isomers of other carotenoids) into less desirable and potentially even harmful isomers.
For these and other reasons, it is believed that microcrystalline carotenoid-in-oil preparations as disclosed herein offer potentially significant advantages over other previously disclosed types of carotenoid preparations, especially during the scale-up of manufacturing operations to create commercial quantities of certain types of carotenoids (notably including zeaxanthin).
The importance of microcrystalline (and ideally, nanoparticle) preparations is discussed in further detail, under the next subheading.
Smaller Particles, Higher Bioavailability
As is well known in the art, most carotenoids (including zeaxanthin) have very low solubility in water; they are strongly hydrophobic. Accordingly, if suspended in an aqueous solution, carotenoids droplets tend to coalesce and/or aggregate, to form larger, relatively sticky masses.
This poses a major difficulty, for carotenoids that are intended to be orally ingested as drugs or nutritional supplements. Orally-ingested carotenoids must be manufactured carefully, to ensure adequate “bioavailability”.
The most common indicator of bioavailability refers to the concentration of a compound which, after oral ingestion by a human or test animal, can be detected as in circulating blood. Accordingly, terms such as “increased” (or improved, enhanced, higher, etc.) bioavailability imply that a certain quantity of an orally-ingested carotenoid will provide higher concentrations of that carotenoid, in circulating blood, if compared to a carotenoid preparation having the same weight, but lower bioavailability. Since deposition of ZX in the macular portion of the retina (at least, in patients whose maculas are suffering from a deficit of ZX) will depend heavily on blood concentrations, it is assumed that any method of generating higher concentrations of ZX in circulating blood, by using formulations with higher levels of bioavailability, will indeed leader to higher quantities will be deposited more rapidly in the retina, in patients in need of such treatment.
Alternately or additionally, zeaxanthin and lutein concentrations in the retina can be measured using any of several non-invasive optical methods, which include flicker photometry (an older and less accurate standard), spectral fundus reflectance, and laser mapping. These measuring techniques are discussed in Berendschot et al, Investig. ophthalm. Vis. Sci. 40: S314, 1999.
Accordingly, when zeaxanthin and/or lutein are involved, actual deposition of the administered carotenoid into the macular portion of the retina (which can then be compared to earlier baseline measurements, for the same human or animal) can also be used as an indicator of greater or lesser bioavailability, for preparations of zeaxanthin or lutein.
A general rule for carotenoids (and other hydrophobic compounds) that are to be orally ingested is this: higher levels of bioavailability will result, if smaller particle sizes are used. This is because, for a given weight of any hydrophobic substance that cannot dissolve in water, smaller particle sizes lead directly to larger exposed surface areas. As a simple numerical example, if you take a cube of material which is 1 millimeter wide, high, and long, and chop it into cubes that are 1 micron on each side, then without changing the weight or volume of that compound at all, you will increase its exposed surface area by a factor of 1000 (from 6 square millimeters, initially provided by a single cube, to 60 square centimeters of surface area, provided by a billion tiny cubes). If that material is made of an oily compound that does not dissolve in water, the increase in exposed surface area will lead to higher bioavailability.
Accordingly, if ZX is put into a microcrystalline formulation, as disclosed herein, that formulation will be more bioavailable than a “coarse-grained” formulation, and it will lead to higher quantities of actual zeaxanthin deposition in the macular portion of the retina, when orally ingested by people who need to increase ZX concentrations inside their retinas.
In reviewing the prior art, it should be recognized that large numbers of prior issued patents disclose various methods of manufacturing various hydrophobic organic compounds. Although some of these processes might be applied to zeaxanthin and other similar carotenoids, they all appear to suffer from various shortcomings and limitations which would render them substantially more expensive (and probably less effective) than the process disclosed herein. In addition, the known prior methods do not appear to be oriented toward creating compounds with the smallest possible particle size, to increase bioavailability. Instead, they appear to be predominantly concerned with creating formulations with improved handling and extended shelf-life, rather than increased bioavailability.
For example, U.S. Pat. No. 5,356,636 (Schneider et al 1994) discloses the use of gelatin, combined with various organic amino compounds, to create dried, powdered formulations of carotenoids and hydrophobic vitamins. U.S. Pat. No. 5,639,441 (Sievers et al, 1997) discloses methods that require the use of “supercritical” carbon dioxide (i.e., a gas which has been placed under extreme pressure, to convert it into a liquid) to generate air-borne aerosols when the solvent mixture is rapidly decompressed. U.S. Pat. No. 4,522,743 (Horn et al, 1985) requires the use of high temperatures and high pressures, the mixing of the carotenoid with a swellable colloid such as gelatin, and the precipitation of the mixture as a “colloidally dispersed” form, using a process commonly known as “flash-cooling”.
Several patents issued to Haynes (including U.S. Pat. Nos. 4,725,442; 5,091,187; 5,091,188; and 5,246,707) require the use of phospholipids to create liposomes that will microencapsulate their contents. U.S. Pat. No. 5,350,773 (Schweikert et al 1994), uses glycerol and compounds such as ascorbyl palmitate to create stabilized “finely dispersed” preparations of a hydrophobic compound such as Vitamin E; however, the creation of the “finely dispersed” compound simply uses a conventional homogenizer. One other patent of lesser interest is U.S. Pat. No. 5,180,747 (Matsuda et al, 1993) which uses encapsulated vitamin preparations. These encapsulated vitamins also include a carotenoid compound in order to protect the vitamin against degradation by light.
None of these previously known methods or preparative steps can perform in the same way as the steps disclosed herein, which are relatively simple, inexpensive, and suited to commercial-scale manufacturing operations.
Accordingly, one object of this invention is to disclose an improved method for manufacturing zeaxanthin (or other carotenoids) in microcrystalline form, in an oily carrier liquid such as a vegetable oil. These microcrystalline preparations (as defined and used herein, this term refers to zeaxanthin crystals or particles having diameters less than about 10 microns, on average; preferably, these diameters are less than about 4 microns on average) have improved bioavailability, compared to other known preparations under the prior art, and the oily carrier provides them with various manufacturing and other advantages.
Another object of this invention is to disclose a process for manufacturing microcrystalline ZX with improved bioavailability, using a relatively simple and inexpensive processing step that is well-suited for commercial-scale manufacturing.
Another object of this invention is to disclose improved ZX formulations which, when orally ingested, provide higher levels of bioavailability than prior known preparations. These formulations are designed to increase the potency and efficacy of ZX in treating or preventing vision problems such as macular degeneration.
Another object of the subject invention is to disclose improved formulations containing zeaxanthin, lutein, or other carotenoids in microcrystalline form, for use as additives in feeds for poultry, salmonid fish, etc. The increased levels of bioavailability of these carotenoid in such formulations allows lower quantities to be used to achieve the desired results, and thereby increases the cost-effectiveness and reduces the total cost of carotenoid additives in animal feeds.
In addition, another object of this invention is to disclose an improved method for commercial-scale manufacturing of other carotenoids which are similar in various respects to zeaxanthin (such as beta-carotene, lycopene, astaxanthin, lutein, etc.), in a microcrystalline form (having particle sizes less than about 4 microns on average) and in an oily carrier liquid, to increase the bioavailability of the microcrystalline carotenoids compared to prior known preparations.
These and other objects of the invention will become more apparent through the following summary, drawings, and description of the preferred embodiments.